Carotenoids represent one of the most widely distributed and structurally diverse classes of natural pigments, producing pigment colors of light yellow to orange to deep red. Eye-catching examples of carotenogenic tissues include carrots, tomatoes, red peppers, and the petals of daffodils and marigolds. Carotenoids are synthesized by all photosynthetic organisms, as well as some bacteria and fungi. These pigments have important functions in photosynthesis, nutrition, and protection against photooxidative damage. For example, animals do not have the ability to synthesize carotenoids but must instead obtain these nutritionally important compounds through their dietary sources. Structurally, carotenoids are 40-carbon (C40) terpenoids derived from the isoprene biosynthetic pathway and its five-carbon universal isoprene building block, isopentenyl pyrophosphate (IPP). This biosynthetic pathway can be divided into two portions: the upper isoprene pathway, which leads to the formation of IPP, and the lower carotenoid biosynthetic pathway, which converts IPP into long C30 and C40 carotenogenic compounds. Both portions of this pathway are shown in FIG. 1.
Various other crt genes are known, which enable the intramolecular conversion of long C30 and C40 compounds to produce numerous other carotenoid compounds. It is the degree of the carbon backbone's unsaturation, conjugation and isomerization which determines the specific carotenoids unique absorption characteristics and colors. Several reviews discuss the genetics of carotenoid pigment biosynthesis, such as those of Armstrong (J. Bact. 176: 4795-4802 (1994); Annu. Rev. Microbiol. 51:629-659 (1997)).
In reference to the availability of carotenoid genes, public domain databases such as GenBank contain sequences isolated from numerous organisms. For example, there are currently 26 GenBank Accession numbers relating to various crtE genes isolated from 19 different organisms. The less frequently encountered crtZ gene boasts 6 GenBank Accession numbers with each gene isolated from a different organism. A similarly wide selection of carotenoid genes is available for each of the genes discussed above.
The genetics of carotenoid pigment biosynthesis has been extremely well studied in the Gram-negative, pigmented bacteria of the genera Pantoea, formerly known as Erwinia. In both E. herbicola EHO-10 (ATCC 39368) and E. uredovora 20D3 (ATCC 19321), the crt genes are clustered in two genetic units, crt Z and crt EXYIB (U.S. Pat. Nos. 5,656,472; 5,5545,816; 5,530,189; 5,530,188; 5,429,939). Despite the similarity in operon structure, the DNA sequences of E. uredovora and E. herbicola show no homology by DNA-DNA hybridization (U.S. Pat. No. 5,429,939).
Although more than 600 different carotenoids have been identified in nature, only a few are used industrially for food colors, animal feeding, pharmaceuticals and cosmetics. Presently, most of the carotenoids used for industrial purposes are produced by chemical synthesis; however, these compounds are very difficult to make chemically (Nelis and Leenheer, Appl. Bacteriol. 70:181-191 (1991)). Natural carotenoids can either be obtained by extraction of plant material or by microbial synthesis. At the present time, only a few plants are widely used for commercial carotenoid production. However, the productivity of carotenoid synthesis in these plants is relatively low and the resulting carotenoids are very expensive.
A number of carotenoids have been produced from microbial sources. For example, Lycopene has been produced from genetically engineered E. coli and Candia utilis (Farmer W. R. and J. C. Liao. (2001) Biotechnol. Prog. 17: 57-61; Wang C. et al., (2000) Biotechnol Prog. 16: 922-926; Misawa, N. and H. Shimada. (1998). J. Biotechnol. 59:169-181; Shimada, H. et al. 1998. Appl. Environm. Microbiol. 64:2676-2680). β-carotene has been produced from E. coli, Candia utilis and Pfaffia rhodozyma (Albrecht, M. et al., (1999). Biotechnol. Lett. 21: 791-795; Miura, Y. et al., 1998. Appl. Environm. Microbiol. 64:1226-1229; U.S. Pat. No. 5,691,190). Zeaxanthin has been produced from recombinant from E. coli and Candia utilis (Albrecht, M. et al., (1999). Biotechnol. Lett. 21: 791-795; Miura, Y. et al., 1998. Appl. Environm. Microbiol. 64:1226-1229). Astaxanthin has been produced from E. coli and Pfaffia rhodozyma (U.S. Pat. Nos. 5,466,599; 6,015,684; 5,182,208; 5,972,642).
Additionally genes encoding various elements of the carotenoid biosynthetic pathway have been cloned and expressed in various microbes. For example genes encoding lycopene cyclase, geranylgeranyl pyrophosphate synthase, and phytoene dehydrogenase isolated from Erwinia herbicola have been expressed recombinantly in E. coli (U.S. Pat. Nos. 5,656,472; 5,545,816; 5,530,189; 5,530,188). Similarly genes encoding the carotenoid products geranylgeranyl pyrophosphate, phytoene, lycopene, β-carotene, and zeaxanthin-diglucoside, isolated from Erwinia uredovora have been expressed in E. coli, Zymomonas mobilis, and Saccharomyces cerevisiae (U.S. Pat. No. 5,429,939). Similarly, the Carotenoid biosynthetic genes crtE (1), crtB (3), crtI (5), crtY (7), and crtZ isolated from Flavobacterium have been recombinantly expressed (U.S. Pat. No. 6,124,113).
Although the above methods of propducing carotenoids are useful, these methods suffer from low yields and reliance on expensive feedstock's. A method that produces higher yields of carotenoids from an inexpensive feedstock is needed.
There are a number of microorganisms that utilize single carbon substrates as sole energy sources. These substrates include, methane, methanol, formate, methylated amines and thiols, and various other reduced carbon compounds which lack any carbon-carbon bonds and are generally quite inexpensive. These organisms are referred to as methylotrophs and herein as “C1 metabolizers”. These organisms are characterized by the ability to use carbon substrates lacking carbon to carbon bonds as a sole source of energy and biomass. A subset of methylotrophs are the methanotrophs which have the unique ability to utilize methane as a sole energy source. Although a large number of these organisms are known, few of these microbes have been successfully harnessed to industrial processes for the synthesis of materials. Although single carbon substrates are cost effective energy sources, difficulty in genetic manipulation of these microorganisms as well as a dearth of information about their genetic machinery has limited their use primarily to the synthesis of native products. For example the commercial applications of biotransformation of methane have historically fallen broadly into three categories: 1) Production of single cell protein, (Sharpe D. H. BioProtein Manufacture 1989. Ellis Horwood series in applied science and industrial technology. New York: Halstead Press.) (Villadsen, John, Recent Trends Chem. React. Eng., [Proc. Int. Chem. React. Eng. Conf.], 2nd (1987), Volume 2, 320-33. Editor(s): Kulkarni, B. D.; Mashelkar, R. A.; Sharma, M. M. Publisher: Wiley East., New Delhi, India; Naguib, M., Proc. OAPEC Symp. Petroprotein, [Pap.] (1980), Meeting Date 1979, 253-77 Publisher: Organ. Arab Pet. Exporting Countries, Kuwait, Kuwait.); 2) epoxidation of alkenes for production of chemicals (U.S. Pat. No. 4,348,476); and 3) biodegradation of chlorinated pollutants (Tsien et al., Gas, Oil, Coal, Environ. Biotechnol. 2, [Pap. Int. IGT Symp. Gas, Oil, Coal, Environ. Biotechnol.], 2nd (1990), 83-104. Editor(s): Akin, Cavit; Smith, Jared. Publisher: Inst. Gas Technol., Chicago, Ill.; WO 9633821; Merkley et al., Biorem. Recalcitrant Org., [Pap. Int. In Situ On-Site Bioreclam. Symp.], 3rd (1995), 165-74. Editor(s): Hinchee, Robert E; Anderson, Daniel B.; Hoeppel, Ronald E. Publisher: Battelle Press, Columbus, Ohio.: Meyer et al., Microb. Releases (1993), 2(1), 11-22). Even here, the commercial success of the methane bio-transformation has been limited to epoxidation of alkenes due to low product yields, toxicity of products and the large amount of cell mass required to generate product associated with the process.
The commercial utility of methylotrophic organisms is reviewed in Lidstrom and Stirling (Annu. Rev. Microbiol. 44:27-58 (1990)). Little commercial success has been documented, despite numerous efforts involving the application of methylotrophic organisms and their enzymes (Lidstrom and Stirling, supra, Table 3). In most cases, it has been discovered that the organisms have little advantage over other well-developed host systems. Methanol is frequently cited as a feedstock which should provide both economic and quality advantages over other more traditional carbohydrate raw materials, but thus far this expectation has not been significantly validated in published works.
One of the most common classes of single carbon metabolizers are the methanotrophs. Methanotrophic bacteria are defined by their ability to use methane as a sole source of carbon and energy. Methane monooxygenase is the enzyme required for the primary step in methane activation and the product of this reaction is methanol (Murrell et al., Arch. Microbiol. (2000), 173(5-6), 325-332). This reaction occurs at ambient temperature and pressures whereas chemical transformation of methane to methanol requires temperatures of hundreds of degrees and high pressure (Grigoryan, E. A., Kinet. Catal. (1999), 40(3), 350-363; WO 2000007718; U.S. Pat. No. 5,750,821). It is this ability to transform methane under ambient conditions along with the abundance of methane that makes the biotransformation of methane a potentially unique and valuable process.
Many methanotrophs contain an inherent isoprenoid pathway which enables these organisms to synthesize other non-endogenous isoprenoid compounds. Since methanotrophs can use one carbon substrate (methane or methanol) as an energy source, it is possible to produce carotenoids at low cost.
Current knowledge in the field concerning methylotrophic organisms and carotenoids leads to the following conclusions. First, there is tremendous commercial incentive arising from abundantly available C1 sources, which could be used as a feedstock for C1 organisms and which should provide both economic and quality advantages over other more traditional carbohydrate raw materials. Secondly, there is abundant knowledge available concerning organisms that possess carotenogenic biosynthetic genes, the function of those genes, and the upper isoprene pathway which produces carotenogenic precursor molecules. Finally, numerous methylotrophic organisms exist in the art which are themselves pigmented, and thereby possess portions of the necessary carotenoid biosynthetic pathway.
Despite these available tools, the art does not reveal any C1 metabolizers which have been genetically engineered to make specific carotenoids of choice, for large scale commercial value. It is hypothesized that the usefulness of these organisms for production of a larger range of chemicals is constrained by limitations including, relatively slow growth rates of methanotrophs, limited ability to tolerate methanol as an alternative substrate to methane, difficulty in genetic engineering, poor understanding of the roles of multiple carbon assimilation pathways present in methanotrophs, and potentially high costs due to the oxygen demand of fully saturated substrates such as methane. The problem to be solved, therefore is to provide a cost effective method for the microbial production of carotenoid compounds, using organisms which utilize C1 compounds as their carbon and energy source.
Applicants have solved the stated problem by engineering microorganisms which are able to use single carbon substrates as sole carbon sources for the production of carotenoid compounds.